Project Summary Asthma is a heterogeneous disease that affects more than 300 million people worldwide, characterized by persistent inflammation and tissue remodeling. The mechanisms underlying chronic inflammation are still not fully understood and patients need better treatments to prevent or revert persistent airway inflammation. Group V phospholipase A2 (Pla2g5) is an enzyme that generates lipid mediators from membrane phospholipids. In the previous award cycle, we demonstrated that Pla2g5 was necessary for the development of mouse and human M2 macrophages, macrophages involved in Type 2 immune responses typical of asthma. We showed that in a mouse model of allergic pulmonary inflammation Pla2g5 was also necessary in macrophages for the recruitment of proinflammatory cells into the lung. Using a model of Alternaria Alternata-induced pulmonary inflammation, which relies on activation of Type 2 innate lymphoid cells (ILC2s), a class of IL-33 responsive cells that generate Th2 cytokines, we found that mice lacking Pla2g5 had impaired ILC2 activation and impaired IL-33 induction by macrophages. Mass spectrometry analysis of lipids showed that Pla2g5 was required in mouse macrophages to release free fatty acids (FFAs), linoleic acid (LA) and oleic acid (OA), which in association with IL-33 amplified ILC2 activation and pulmonary inflammation. The goal of this application is to investigate the mechanisms whereby IL-33 and FFAs, generated by macrophages expressing Pla2g5, perpetuate allergic pulmonary inflammation, their cellular targets and pathways involved. To reach this goal, we will assess the mechanism/s whereby Pla2g5 in macrophages induces IL-33 production, and the role of IL-33 specifically generated by macrophages in allergic pulmonary inflammation. We will use mice that lack Pla2g5, IL-33, and ST2, the receptor for IL-33, specifically in macrophages. To study the function of LA and OA in establishment and perpetuation of the inflammatory response, we will use a model of Alternaria-induced pulmonary inflammation and mice lacking the receptor for LA and OA (FFA receptor-1 [FFAR1]). We will use FFAR1 inhibitors and knocking-down strategies to validate our findings in human cells. These studies will reveal novel innate immune pathways involving macrophages and target immune cells, which are likely mediated by FFAs and IL33 and are responsible for the persistent airway inflammation that is characteristic of asthmatic patients. The proposed studies should lend substantial new insight with strong potential for new therapeutic applications for prevention and treatment of persistent inflammation typical of asthmatic patients.